HER3 specific monoclonal antibodies for diagnostic and therapeutic use

ABSTRACT

Isolated or recombinant anti-HER3 monoclonal antibodies are provided. In some cases, antibodies of the embodiments can be used for the detection, diagnosis and/or therapeutic treatment of human diseases, such as cancer.

This application is a continuation of U.S. patent application Ser. No.15/643,690, filed Jul. 7, 2017, which is a division of U.S. patentapplication Ser. No. 14/774,808, filed Sep. 11, 2015, as a nationalphase application under 35 U.S.C. § 371 of International Application No.PCT/US2014/025,446, filed Mar. 13, 2014, which claims the benefit ofU.S. Provisional Patent Application No. 61/782,770, filed Mar. 14, 2013,the entirety of each of which is incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UTSHP0295USC1_ST25.txt”, which is 25 KB (as measured in MicrosoftWindows®) and was created on Jun. 12, 2019, is filed herewith byelectronic submission and is incorporated by reference herein.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of cancer biology.More particularly, it concerns HER3 targeting monoclonal antibodies forthe treatment and detection of cancer.

2. Description of Related Art

The epidermal growth factor receptor (EGFR) family (ErbB/HER) consistsof four known members: EGFR (HER1, erbB-1), HER2 (erbB-2), HER3(erbB-3), and HER4 (erbB-4). Each receptor protein has the same basicstructure, consisting of an extracellular amino-terminal domain, asingle transmembrane spanning sequence and an intracellular cytoplasmicdomain. ErbB signaling has a complex network with more than 11interacting ligands for distinct binding specificities and activation ofsignaling pathways. The complex contents and interactions of HERreceptors and ligands provide a great potential for significant signaldiversification.

Growing evidence indicates that HER3 plays important role in theresistance mechanisms of HER targeting therapeutics including both smallmolecule tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotiniband lapatinib, and HER family receptor targeting monoclonal antibodies,such as trastuzumab, cetuximab, panitumumab and pertuzumab.Heregulin/Neuregulin (NRG) is a member of a complex ligand familyinteracting with HER3 and HER4. Neuregulin binding activates ErbB3 andleads to formation of heterodimeric receptor complexes and activation ofdown stream signaling of HER3 through both PI3K/AKT and Ras/Raf/MAPKpathways. Therefore, HER3-binding monoclonal antibodies to blockneuregulin binding have the potential to block HER3 signaling andinhibit cancer cell proliferation.

SUMMARY OF THE INVENTION

Described herein are HER3 monoclonal antibodies that potently block HER3signaling and inhibit cancer cell proliferation. Thus, in a firstembodiment, there is provided an isolated or recombinant monoclonalantibody that specifically binds to a HER3. In certain aspects, anantibody competes for the binding of a HER3 with the Rab46, Rab1210,Rab189, or Rab774 monoclonal antibody. In certain aspects, the antibodymay comprise all or part of the heavy chain variable region and/or lightchain variable region of the Rab46, Rab1210, Rab189, or Rab774monoclonal antibodies. In a further aspect, the antibody may comprise anamino acid sequence that corresponds to a first, second, and/or thirdcomplementarity determining region (CDR) from the light variable and/orheavy variable chain of the Rab46, Rab1210, Rab189, or Rab774 monoclonalantibodies of the present embodiments.

In certain aspects, the isolated antibody comprises CDR sequences atleast 80%, 90%, or 95% identical to the CDR regions of the Rab46,Rab1210, Rab189, or Rab774 heavye and light chain amino acid sequences.In further aspects, an antibody comprises CDR regions identical to theRab46, Rab1210, Rab189, or Rab774 CDR regions, except for one or twoamino acid substitutions, deletions, or insertions at one or more of theCDRs. For example, the antibody can comprise CDRs wherein the CDRsequences comprise 1 or 2 amino acid substitutions in the V_(H) CDR1,V_(H) CDR2, V_(H) CDR3, V_(L) CDR1, V_(L) CDR2 and/or V_(L) CDR3relative to the CDRs of a Rab46, Rab1210, Rab189, or Rab774 monoclonalantibody. Thus, in some specific aspects, an antibody of the embodimentscomprises (a) a first V_(H) CDR at least 80% identical to V_(H) CDR1 ofRab46 (SEQ ID NO: 9), Rab1210 (SEQ ID NO: 15), Rab189 (SEQ ID NO: 21),or Rab774 (SEQ ID NO: 27); (b) a second V_(H) CDR at least 80% identicalto V_(H) CDR2 of Rab46 (SEQ ID NO: 10), Rab1210 (SEQ ID NO: 16), Rab189(SEQ ID NO: 22), or Rab774 (SEQ ID NO: 28); (c) a third V_(H) CDR atleast 80% identical to V_(H) CDR3 of Rab46 (SEQ ID NO: 11), Rab1210 (SEQID NO: 17), Rab189 (SEQ ID NO: 23), or Rab774 (SEQ ID NO: 29); (d) afirst V_(L) CDR at least 80% identical to V_(L) CDR1 of Rab46 (SEQ IDNO: 12), Rab1210 (SEQ ID NO: 18), Rab189 (SEQ ID NO: 24), or Rab774 (SEQID NO: 30); (e) a second V_(L) CDR at least 80% identical to V_(L) CDR2of Rab46 (SEQ ID NO: 13), Rab1210 (SEQ ID NO: 19), Rab189 (SEQ ID NO:25), or Rab774 (SEQ ID NO: 31); and (f) a third V_(L) CDR at least 80%identical to V_(L) CDR3 of Rab46 (SEQ ID NO: 14), Rab1210 (SEQ ID NO:20), Rab189 (SEQ ID NO: 26), or Rab774 (SEQ ID NO: 32). In certainaspects, such an antibody is a humanized or de-immunized antibodycomprising the foregoing CDRs on a human IgGs (e.g., IgG1, IgG2, IgG4,or a genetically modified IgG) backbone.

In further aspects, the isolated antibody comprises a first V_(H), asecond V_(H), a third V_(H), a first V_(L), a second V_(L), and a thirdV_(L) CDR sequence at least 80% identical to the corresponding CDRsequence of monoclonal antibody Rab46, which are represented by SEQ IDNOs: 9, 10, 11, 12, 13, and 14, respectively. In one aspect, theisolated antibody comprises CDR sequences that are identical to the CDRsequences of monoclonal antibody Rab46.

In another aspect, the isolated antibody comprises a V_(H) domain atleast about 80% identical to the V_(H) domain of Rab46 (SEQ ID NO: 1);hRab46H-1 (SEQ ID NO: 33); or hRab46H-2 (SEQ ID NO: 34); and a V_(L)domain at least about 80% identical to the V_(L) domain of Rab46 (SEQ IDNO: 2); hRab46L-1 (SEQ ID NO: 35); or hRab46L-2 (SEQ ID NO: 36). Forexample, the antibody can comprise a V_(H) domain at least 95% identicalto the V_(H) domain of hRab46H-1 (SEQ ID NO: 33) and a V_(L) domain atleast 95% identical to the V_(L) domain of hRab46L-1 (SEQ ID NO: 35) orhRab46L-2 (SEQ ID NO: 36). Thus, in some aspects, an antibody comprisesa V_(H) domain identical to the V_(H) domain of hRab46H-1 (SEQ ID NO:33) and a V_(L) domain identical to the V_(L) domain of hRab46L-1 (SEQID NO: 35). In further aspects an antibody comprises a V_(H) domainidentical to the V_(H) domain of hRab46H-1 (SEQ ID NO: 33) and a V_(L)domain identical to the V_(L) domain hRab46L-2 (SEQ ID NO: 36). Inanother aspects an antibody comprises a V_(H) domain at least 95%identical to the V_(H) domain of hRab46H-2 (SEQ ID NO: 34) and a V_(L)domain at least 95% identical to the V_(L) domain of hRab46L-2 (SEQ IDNO: 36) or hRab46L-1 (SEQ ID NO: 35). For instance the antibody cancomprise a V_(H) domain identical to the V_(H) domain of hRab46H-2 (SEQID NO: 34) and a V_(L) domain identical to the V_(L) domain of hRab46L-2(SEQ ID NO: 36) or a V_(H) domain identical to the V_(H) domain ofhRab46H-2 (SEQ ID NO: 34) and a V_(L) domain identical to the V_(L)domain of hRab46L-1 (SEQ ID NO: 35). In a specific example, the isolatedantibody can comprise V_(H) and V_(L) domains identical to those ofmonoclonal antibody Rab46, HER3-hMab-A9, HER3-hMab-A10, HER3-hMab-A11,or HER3-hMab-A12. In still further aspects, the antibody is theHER3-hMab-A9, HER3-hMab-A10, HER3-hMab-A11, or HER3-hMab-A12 antibody.

In further aspects, the isolated antibody comprises a first V_(H), asecond V_(H), a third V_(H), a first V_(L), a second V_(L), and a thirdV_(L) CDR sequence at least 80% identical to the corresponding CDRsequence of monoclonal antibody Rab1210, which are represented by SEQ IDNOs: 15, 16, 17, 18, 19, and 20, respectively. In one aspect, theisolated antibody comprises CDR sequences that are identical to the CDRsequences of monoclonal antibody Rab1210.

In another aspect, the isolated antibody comprises a V_(H) domain atleast about 80% identical to the V_(H) domain of Rab1210 (SEQ ID NO: 3);hRab1210H-1 (SEQ ID NO: 37); or hRab1210H-2 (SEQ ID NO: 38); and a V_(L)domain at least about 80% identical to the V_(L) domain of Rab1210 (SEQID NO: 4); hRab1210L-1 (SEQ ID NO: 39); or hRab1210L-2 (SEQ ID NO: 40).Thus, in some aspects, the antibody comprises a V_(H) domain at least95% identical to the V_(H) domain of hRab1210H-1 (SEQ ID NO: 37) and aV_(L) domain at least 95% identical to the V_(L) domain of hRab1210L-1(SEQ ID NO: 39) or hRab1210L-2 (SEQ ID NO: 40). For instance theantibody can comprise a V_(H) domain identical to the V_(H) domain ofhRab1210H-1 (SEQ ID NO: 37) and a V_(L) domain identical to the V_(L)domain of hRab1210L-1 (SEQ ID NO: 39) or a V_(H) domain identical to theV_(H) domain of hRab1210H-1 (SEQ ID NO: 37) and a V_(L) domain identicalto the V_(L) domain of hRab1210L-2 (SEQ ID NO: 40). In further aspects,an antibody comprises a V_(H) domain at least 95% identical to the V_(H)domain of hRab1210H-2 (SEQ ID NO: 38) and a V_(L) domain at least 95%identical to the V_(L) domain of hRab1210L-2 (SEQ ID NO: 40) orhRab1210L-1 (SEQ ID NO: 39). For example, the antibody can comprise aV_(H) domain identical to the V_(H) domain of hRab1210H-2 (SEQ ID NO:38) and a V_(L) domain identical to the V_(L) domain of hRab1210L-2 (SEQID NO: 40) or a V_(H) domain identical to the V_(H) domain ofhRab1210H-2 (SEQ ID NO: 38) and a V_(L) domain identical to the V_(L)domain of hRab1210L-1 (SEQ ID NO: 39). In a specific example, theisolated antibody can comprise V_(H) and V_(L) domains identical tothose of monoclonal antibody Rab1210, HER3-hMab-A13, HER3-hMab-A14,HER3-hMab-A15 or HER3-hMab-A16. In still further aspects, the antibodyis the HER3-hMab-A13, HER3-hMab-A14, HER3-hMab-A15 or HER3-hMab-A16antibody.

In further aspects, the isolated antibody comprises a first V_(H), asecond V_(H), a third V_(H), a first V_(L), a second V_(L), and a thirdV_(L) CDR sequence at least 80% identical to the corresponding CDRsequence of monoclonal antibody Rab189, which are represented by SEQ IDNOs: 21, 22, 23, 24, 25, and 26, respectively. In one aspect, theisolated antibody comprises CDR sequences that are identical to the CDRsequences of monoclonal antibody Rab189.

In another aspect, the isolated antibody comprises a V_(H) domain atleast about 80% identical to the V_(H) domain of Rab189 (SEQ ID NO: 5)and a V_(L) domain at least about 80% identical to the V_(L) domain ofRab189 (SEQ ID NO: 6). In one aspect, the isolated antibody comprisesV_(H) and V_(L) domains identical to those of monoclonal antibodyRab189.

In further aspects, the isolated antibody comprises a first V_(H), asecond V_(H), a third V_(H), a first V_(L), a second V_(L), and a thirdV_(L) CDR sequence at least 80% identical to the corresponding CDRsequence of monoclonal antibody Rab774, which are represented by SEQ IDNOs: 27, 28, 29, 30, 31, and 32, respectively. In one aspect, theisolated antibody comprises CDR sequences that are identical to the CDRsequences of monoclonal antibody Rab774.

In another aspect, the isolated antibody comprises a V_(H) domain atleast about 80% identical to the V_(H) domain of Rab774 (SEQ ID NO: 7)and a V_(L) domain at least about 80% identical to the V_(L) domain ofRab774 (SEQ ID NO: 8). In one aspect, the isolated antibody comprisesV_(H) and V_(L) domains identical to those of monoclonal antibodyRab774.

In some aspects, an antibody of the embodiments may be an IgG (e.g.,IgG1, IgG2, IgG3 or IgG4), IgM, IgA, genetically modified IgG isotype,or an antigen binding fragment thereof. The antibody may be a Fab′, aF(ab′)2 a F(ab′)3, a monovalent scFv, a bivalent scFv, a bispecific or asingle domain antibody. The antibody may be a human, humanized, orde-immunized antibody. In a further aspect, the isolated antibody is theRab46, Rab1210, Rab189, or Rab774 antibody.

In some aspects, the antibody may be conjugated to an imaging agent, achemotherapeutic agent, a toxin, or a radionuclide.

In one embodiment, there is provided a recombinant polypeptidecomprising an antibody V_(H) domain comprising CDRs 1-3 of the V_(H)domain of Rab46 (SEQ ID NOs: 9, 10, and 11); CDRs 1-3 of the V_(H)domain of Rab1210 (SEQ ID NOs: 15, 16, and 17); CDRs 1-3 of the V_(H)domain of Rab189 (SEQ ID NOs: 21, 22, and 23); or CDRs 1-3 of the V_(H)domain of Rab774 (SEQ ID NOs: 27, 28, and 29). In another embodiment,there is provided a recombinant polypeptide comprising an antibody V_(L)domain comprising CDRs 1-3 of the V_(L) domain of Rab46 (SEQ ID NOs: 12,13, and 14); Rab1210 (SEQ ID NOs: 18, 19, and 20); Rab189 (SEQ ID NOs:24, 25, and 26); or Rab774 (SEQ ID NOs: 30, 31, and 32).

In some embodiments, there is provided an isolated polynucleotidemolecule comprising nucleic acid sequence encoding an antibody or apolypeptide comprising an antibody V_(H) or V_(L) domain disclosedherein.

In further embodiments, a host cell is provided that produces amonoclonal antibody or recombinant polypeptide of the embodiments. Insome aspects, the host cell is a mammalian cell, a yeast cell, abacterial cell, a ciliate cell, or an insect cell. In certain aspects,the host cell is a hybridoma cell.

In still further embodiments, there is provided a method ofmanufacturing an antibody of the present invention comprising expressingone or more polynucleotide molecule(s) encoding a V_(L) or V_(H) chainof an antibody disclosed herein in a cell and purifying the antibodyfrom the cell.

In additional embodiments, there are pharmaceutical compositionscomprising an antibody or antibody fragment as discussed herein. Such acomposition further comprises a pharmaceutically acceptable carrier andmay or may not contain additional active ingredients.

In embodiments of the present invention, there is provided a method fortreating a subject having a cancer comprising administering an effectiveamount of an antibody disclosed herein. In certain aspects, the antibodyis a monoclonal antibody of the present invention, such as the Rab46,Rab1210, Rab189, Rab774, HER3-hMab-A9, HER3-hMab-A10, HER3-hMab-A11,HER3-hMab-A12, HER3-hMab-A13, HER3-hMab-A14, HER3-hMab-A15 orHER3-hMab-A16 antibody or a recombinant polypeptide comprising antibodysegment derived therefrom.

In certain aspects, the cancer may be a breast cancer, lung cancer, head& neck cancer, prostate cancer, esophageal cancer, tracheal cancer,brain cancer, liver cancer, bladder cancer, stomach cancer, pancreaticcancer, ovarian cancer, uterine cancer, cervical cancer, testicularcancer, colon cancer, rectal cancer or skin cancer.

In one aspect, the antibody may be administered systemically. Inadditional aspects, the antibody may be administered intravenously,intradermally, intratumorally, intramuscularly, intraperitoneally,subcutaneously, or locally. The method may further compriseadministering at least a second anticancer therapy to the subject.Examples of the second anticancer therapy include, but are not limitedto, surgical therapy, chemotherapy, radiation therapy, cryotherapy,hormonal therapy, immunotherapy, or cytokine therapy.

In further aspects, the method may further comprise administering acomposition of the present invention more than one time to the subject,such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or moretimes.

In another embodiment, there is provided a method for detecting a cancerin a subject comprising testing for the presence of elevated HER3relative to a control in a sample from the subject, wherein the testingcomprises contacting the sample with an antibody disclosed herein. Forexample, the method may be an in vitro or in vivo method.

Certain embodiments are directed to an antibody or recombinantpolypeptide composition comprising an isolated and/or recombinantantibody or polypeptide that specifically binds HER3. In certain aspectsthe antibody or polypeptide has a sequence that is, is at least, or isat most 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical (or any rangederivable therein) to all or part of any monoclonal antibody providedherein. In still further aspects the isolated and/or recombinantantibody or polypeptide has, has at least, or has at most 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or morecontiguous amino acids from any of the sequences provided herein or acombination of such sequences.

In still further aspects, an antibody or polypeptide of the embodimentscomprises one or more amino acid segments of the any of the amino acidsequences disclosed herein. For example, the antibody or polypeptide cancomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid segmentscomprising about, at least or at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or200 amino acids in length, including all values and ranges therebetween, that are at least 80, 85, 90, 95, 96, 97, 98, 99, or 100%identical to any of the amino acid sequences disclosed herein. Incertain aspects the amino segment(s) are selected from one of the aminoacid sequences of a HER3-binding antibody as provided in Table 6 orChart 1.

In still further aspects, an antibody or polypeptide of the embodimentscomprises an amino acid segment of the any of the amino acid sequencesdisclosed herein, wherein the segment begins at amino acid position 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, or 200 in any sequence providedherein and ends at amino acid position 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, or 200 in the same provided sequence. In certain aspects the aminosegment(s), or portions thereof, are selected from one of the amino acidsequences of a HER3-binding antibody as provided in Table 6 or Chart 1.

In yet further aspects, an antibody or polypeptide of the embodimentscomprises an amino acid segment that is at least 80, 85, 90, 95, 96, 97,98, 99, or 100% identical (or any range derivable therein) to a V, VJ,VDJ, D, DJ, J or CDR domain of a HER3-binding antibody (as provided inTable 6 and Chart 1). For example, a polypeptide may comprise 1, 2 or 3amino acid segment that are at least 80, 85, 90, 95, 96, 97, 98, 99, or100% identical (or any range derivable therein) to CDRs 1, 2, and/or 3 aHER3-binding antibody as provided in Table 6 and Chart 1.

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Process flow for antibody selection.

FIG. 2. Phage display vector for Rabbit Fab expression.

FIG. 3. Binding of anti-Her3 antibodies on human and mouse HER3/ErbB3using heterologous HER3 expressing CHO cells.

FIG. 4. Dose response of purified anti-Her3 IgGs in inhibition of HER3phosphorylation assay.

FIG. 5. pAKT and pERK inhibition by HER3 Mab IgGs in MCF7 cells.

FIG. 6. Ligand blocking assay using alpha screening format and graphsshow dose response of anti-HER3 antibodies.

FIG. 7. Inhibition of NRG-induced cell growth in MCF7 cells by anti HER3antibodies. Data are shown as percentage of reduction by the antibodiesrelative to no antibody control.

FIGS. 8A-B. Concentration dependent binding of the humanized HER3monoclonal antibodies on HER3 ECD by ELISA.

FIG. 9. Kinetic binding affinity of the humanized HER3 monoclonalantibodies on HER3 ECD by Biacore assay.

FIG. 10. Concentration dependent inhibition of pHER3 by the humanizedHER3 monoclonal antibodies in T47D cancer cells.

FIG. 11. Concentration dependent inhibition of CWR22 cancer cellproliferation by the humanized HER3 monoclonal antibodies.

FIG. 12. Concentration dependent inhibition of MCF-7 cancer cellproliferation by the humanized HER3 monoclonal antibodies.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. Antibodies of the Invention

In certain embodiments, an antibody or a fragment thereof that binds toat least a portion of HER3 protein and inhibits HER3 signaling andcancer cell proliferation are contemplated. As used herein, the term“antibody” is intended to refer broadly to any immunologic bindingagent, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG aswell as polypeptides comprising antibody CDR domains that retain antigenbinding activity. The antibody may be selected from the group consistingof a chimeric antibody, an affinity matured antibody, a polyclonalantibody, a monoclonal antibody, a humanized antibody, a human antibody,or an antigen-binding antibody fragment or a natural or syntheticligand.

Preferably, the anti-HER3 antibody is a monoclonal antibody or ahumanized antibody. Indeed studies presented herein demonstrated thatthe identified CDRs for HER3-binding antibodies could be placed in ahuman frame work and the resulting humanized antibodies retained highaffinity for HER3 (FIG. 8-9). Importantly the humanized antibodies alsoefficiently inhibited HER3 signaling (FIG. 10) and were able to inhibitcancer cell replication in a HER3 positive cancer cells (FIGS. 11-12).Thus, the HER3-binding antibodies provided herein, in particular thehumanized antibodies, are promising candidates for new anticancertherapeutics.

Thus, by known means and as described herein, polyclonal or monoclonalantibodies, antibody fragments, and binding domains and CDRs (includingengineered forms of any of the foregoing) may be created that arespecific to HER3 protein, one or more of its respective epitopes, orconjugates of any of the foregoing, whether such antigens or epitopesare isolated from natural sources or are synthetic derivatives orvariants of the natural compounds. Another variation is the constructionof bispecific antibodies in which one heavy chain targeting HER3 andother heavy chain targeting a different cancer target, such as HER2,EGFR, IGF1R, cMet, or other cell surface targets.

Examples of antibody fragments suitable for the present embodimentsinclude, without limitation: (i) the Fab fragment, consisting of V_(L),V_(H), C_(L), and C_(H1) domains; (ii) the “Fd” fragment consisting ofthe V_(H) and C_(H1) domains; (iii) the “Fv” fragment consisting of theV_(L) and V_(H) domains of a single antibody; (iv) the “dAb” fragment,which consists of a V_(H) domain; (v) isolated CDR regions; (vi) F(ab′)2fragments, a bivalent fragment comprising two linked Fab fragments;(vii) single chain Fv molecules (“scFv”), wherein a V_(H) domain and aV_(L) domain are linked by a peptide linker that allows the two domainsto associate to form a binding domain; (viii) bi-specific single chainFv dimers (see U.S. Pat. No. 5,091,513); and (ix) diabodies, multivalentor multispecific fragments constructed by gene fusion (US Patent App.Pub. 20050214860). Fv, scFv, or diabody molecules may be stabilized bythe incorporation of disulphide bridges linking the V_(H) and V_(L)domains. Minibodies comprising a scFv joined to a CH3 domain may also bemade (Hu et al., 1996).

Antibody-like binding peptidomimetics are also contemplated inembodiments. Liu et al. (2003) describe “antibody like bindingpeptidomimetics” (ABiPs), which are peptides that act as pared-downantibodies and have certain advantages of longer serum half-life as wellas less cumbersome synthesis methods.

Animals may be inoculated with an antigen, such as a HER3 extracellulardomain protein (amino acids 1-643 of NCBI Accession No. M34309), inorder to produce antibodies specific for HER3 protein. Frequently anantigen is bound or conjugated to another molecule to enhance the immuneresponse. As used herein, a conjugate is any peptide, polypeptide,protein, or non-proteinaceous substance bound to an antigen that is usedto elicit an immune response in an animal. Antibodies produced in ananimal in response to antigen inoculation comprise a variety ofnon-identical molecules (polyclonal antibodies) made from a variety ofindividual antibody producing B lymphocytes. A polyclonal antibody is amixed population of antibody species, each of which may recognize adifferent epitope on the same antigen. Given the correct conditions forpolyclonal antibody production in an animal, most of the antibodies inthe animal's serum will recognize the collective epitopes on theantigenic compound to which the animal has been immunized. Thisspecificity is further enhanced by affinity purification to select onlythose antibodies that recognize the antigen or epitope of interest.

A monoclonal antibody is a single species of antibody wherein everyantibody molecule recognizes the same epitope because all antibodyproducing cells are derived from a single B-lymphocyte cell line. Themethods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies. Insome embodiments, rodents such as mice and rats are used in generatingmonoclonal antibodies. In some embodiments, rabbit, sheep, or frog cellsare used in generating monoclonal antibodies. The use of rats is wellknown and may provide certain advantages. Mice (e.g., BALB/c mice) areroutinely used and generally give a high percentage of stable fusions.

Hybridoma technology involves the fusion of a single B lymphocyte from amouse previously immunized with a HER3 antigen with an immortal myelomacell (usually mouse myeloma). This technology provides a method topropagate a single antibody-producing cell for an indefinite number ofgenerations, such that unlimited quantities of structurally identicalantibodies having the same antigen or epitope specificity (monoclonalantibodies) may be produced.

Plasma B cells may be isolated from freshly prepared rabbit peripheralblood mononuclear cells of immunized rabbits and further selected forHER3 binding cells. After enrichment of antibody producing B cells,total RNA may be isolated and cDNA synthesized. DNA sequences ofantibody variable regions from both heavy chains and light chains may beamplified, constructed into a phage display Fab expression vector, andtransformed into E. coli. HER3 specific binding Fab may be selected outthrough multiple rounds enrichment panning and sequenced. Selected HER3binding hits may be expressed as full length IgG in rabbit andrabbit/human chimeric forms using a mammalian expression vector systemin human embryonic kidney (HEK293) cells (Invitrogen) and purified usinga protein G resin with a fast protein liquid chromatography (FPLC)separation unit.

In one embodiment, the antibody is a chimeric antibody, for example, anantibody comprising antigen binding sequences from a non-human donorgrafted to a heterologous non-human, human, or humanized sequence (e.g.,framework and/or constant domain sequences). Methods have been developedto replace light and heavy chain constant domains of the monoclonalantibody with analogous domains of human origin, leaving the variableregions of the foreign antibody intact. Alternatively, “fully human”monoclonal antibodies are produced in mice transgenic for humanimmunoglobulin genes. Methods have also been developed to convertvariable domains of monoclonal antibodies to more human form byrecombinantly constructing antibody variable domains having both rodent,for example, mouse, and human amino acid sequences. In “humanized”monoclonal antibodies, only the hypervariable CDR is derived from mousemonoclonal antibodies, and the framework and constant regions arederived from human amino acid sequences (see U.S. Pat. Nos. 5,091,513and 6,881,557). It is thought that replacing amino acid sequences in theantibody that are characteristic of rodents with amino acid sequencesfound in the corresponding position of human antibodies will reduce thelikelihood of adverse immune reaction during therapeutic use. Ahybridoma or other cell producing an antibody may also be subject togenetic mutation or other changes, which may or may not alter thebinding specificity of antibodies produced by the hybridoma.

Methods for producing polyclonal antibodies in various animal species,as well as for producing monoclonal antibodies of various types,including humanized, chimeric, and fully human, are well known in theart and highly predictable. For example, the following U.S. patents andpatent applications provide enabling descriptions of such methods: U.S.Patent Application Nos. 2004/0126828 and 2002/0172677; and U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,196,265; 4,275,149;4,277,437; 4,366,241; 4,469,797; 4,472,509; 4,606,855; 4,703,003;4,742,159; 4,767,720; 4,816,567; 4,867,973; 4,938,948; 4,946,778;5,021,236; 5,164,296; 5,196,066; 5,223,409; 5,403,484; 5,420,253;5,565,332; 5,571,698; 5,627,052; 5,656,434; 5,770,376; 5,789,208;5,821,337; 5,844,091; 5,858,657; 5,861,155; 5,871,907; 5,969,108;6,054,297; 6,165,464; 6,365,157; 6,406,867; 6,709,659; 6,709,873;6,753,407; 6,814,965; 6,849,259; 6,861,572; 6,875,434; and 6,891,024.All patents, patent application publications, and other publicationscited herein and therein are hereby incorporated by reference in thepresent application.

Antibodies may be produced from any animal source, including birds andmammals. Preferably, the antibodies are ovine, murine (e.g., mouse andrat), rabbit, goat, guinea pig, camel, horse, or chicken. In addition,newer technology permits the development of and screening for humanantibodies from human combinatorial antibody libraries. For example,bacteriophage antibody expression technology allows specific antibodiesto be produced in the absence of animal immunization, as described inU.S. Pat. No. 6,946,546, which is incorporated herein by reference.These techniques are further described in: Marks (1992); Stemmer (1994);Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996).

It is fully expected that antibodies to HER3 will have the ability toneutralize or counteract the effects of HER3 regardless of the animalspecies, monoclonal cell line, or other source of the antibody. Certainanimal species may be less preferable for generating therapeuticantibodies because they may be more likely to cause allergic responsedue to activation of the complement system through the “Fc” portion ofthe antibody. However, whole antibodies may be enzymatically digestedinto “Fc” (complement binding) fragment, and into antibody fragmentshaving the binding domain or CDR. Removal of the Fc portion reduces thelikelihood that the antigen antibody fragment will elicit an undesirableimmunological response, and thus, antibodies without Fc may bepreferential for prophylactic or therapeutic treatments. As describedabove, antibodies may also be constructed so as to be chimeric orpartially or fully human, so as to reduce or eliminate the adverseimmunological consequences resulting from administering to an animal anantibody that has been produced in, or has sequences from, otherspecies.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of the polypeptide isaffected. Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

Proteins may be recombinant, or synthesized in vitro. Alternatively, anon-recombinant or recombinant protein may be isolated from bacteria. Itis also contemplated that a bacteria containing such a variant may beimplemented in compositions and methods. Consequently, a protein neednot be isolated.

It is contemplated that in compositions there is between about 0.001 mgand about 10 mg of total polypeptide, peptide, and/or protein per ml.Thus, the concentration of protein in a composition can be about, atleast about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or anyrange derivable therein). Of this, about, at least about, or at mostabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% may be an antibody that bindsHER3.

An antibody or preferably an immunological portion of an antibody, canbe chemically conjugated to, or expressed as, a fusion protein withother proteins. For purposes of this specification and the accompanyingclaims, all such fused proteins are included in the definition ofantibodies or an immunological portion of an antibody.

Embodiments provide antibodies and antibody-like molecules against HER3,polypeptides and peptides that are linked to at least one agent to forman antibody conjugate or payload. In order to increase the efficacy ofantibody molecules as diagnostic or therapeutic agents, it isconventional to link or covalently bind or complex at least one desiredmolecule or moiety. Such a molecule or moiety may be, but is not limitedto, at least one effector or reporter molecule. Effector moleculescomprise molecules having a desired activity, e.g., cytotoxic activity.Non-limiting examples of effector molecules that have been attached toantibodies include toxins, therapeutic enzymes, antibiotics,radio-labeled nucleotides and the like. By contrast, a reporter moleculeis defined as any moiety that may be detected using an assay.Non-limiting examples of reporter molecules that have been conjugated toantibodies include enzymes, radiolabel s, haptens, fluorescent labels,phosphorescent molecules, chemiluminescent molecules, chromophores,luminescent molecules, photoaffinity molecules, colored particles orligands, such as biotin.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3-6?-diphenylglycouril-3 attached to the antibody.Monoclonal antibodies may also be reacted with an enzyme in the presenceof a coupling agent such as glutaraldehyde or periodate. Conjugates withfluorescein markers are prepared in the presence of these couplingagents or by reaction with an isothiocyanate.

II. Treatment of Diseases

Certain aspects of the present embodiments can be used to prevent ortreat a disease or disorder associated with HER3 signaling. Signaling ofHER3 may be reduced by any suitable drugs to prevent cancer cellproliferation. Preferably, such substances would be an anti-HER3antibody.

“Treatment” and “treating” refer to administration or application of atherapeutic agent to a subject or performance of a procedure or modalityon a subject for the purpose of obtaining a therapeutic benefit of adisease or health-related condition. For example, a treatment mayinclude administration of a pharmaceutically effective amount of anantibody that inhibits the HER3 signaling.

“Subject” and “patient” refer to either a human or non-human, such asprimates, mammals, and vertebrates. In particular embodiments, thesubject is a human.

The term “therapeutic benefit” or “therapeutically effective” as usedthroughout this application refers to anything that promotes or enhancesthe well-being of the subject with respect to the medical treatment ofthis condition. This includes, but is not limited to, a reduction in thefrequency or severity of the signs or symptoms of a disease. Forexample, treatment of cancer may involve, for example, a reduction inthe size of a tumor, a reduction in the invasiveness of a tumor,reduction in the growth rate of the cancer, or prevention of metastasis.Treatment of cancer may also refer to prolonging survival of a subjectwith cancer.

A. Pharmaceutical Preparations

Where clinical application of a therapeutic composition containing aninhibitory antibody is undertaken, it will generally be beneficial toprepare a pharmaceutical or therapeutic composition appropriate for theintended application. In certain embodiments, pharmaceuticalcompositions may comprise, for example, at least about 0.1% of an activecompound. In other embodiments, an active compound may comprise betweenabout 2% to about 75% of the weight of the unit, or between about 25% toabout 60%, for example, and any range derivable therein.

The therapeutic compositions of the present embodiments areadvantageously administered in the form of injectable compositionseither as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. These preparations also may be emulsified.

The phrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic, or other untoward reaction when administered to an animal,such as a human, as appropriate. The preparation of a pharmaceuticalcomposition comprising an antibody or additional active ingredient willbe known to those of skill in the art in light of the presentdisclosure. Moreover, for animal (e.g., human) administration, it willbe understood that preparations should meet sterility, pyrogenicity,general safety, and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall aqueous solvents (e.g., water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles, such as sodium chloride, Ringer'sdextrose, etc.), non-aqueous solvents (e.g., propylene glycol,polyethylene glycol, vegetable oil, and injectable organic esters, suchas ethyloleate), dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial or antifungal agents, anti-oxidants,chelating agents, and inert gases), isotonic agents, absorption delayingagents, salts, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, fluid and nutrient replenishers, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart. The pH and exact concentration of the various components in apharmaceutical composition are adjusted according to well-knownparameters.

The term “unit dose” or “dosage” refers to physically discrete unitssuitable for use in a subject, each unit containing a predeterminedquantity of the therapeutic composition calculated to produce thedesired responses discussed above in association with itsadministration, i.e., the appropriate route and treatment regimen. Thequantity to be administered, both according to number of treatments andunit dose, depends on the effect desired. The actual dosage amount of acomposition of the present embodiments administered to a patient orsubject can be determined by physical and physiological factors, such asbody weight, the age, health, and sex of the subject, the type ofdisease being treated, the extent of disease penetration, previous orconcurrent therapeutic interventions, idiopathy of the patient, theroute of administration, and the potency, stability, and toxicity of theparticular therapeutic substance. For example, a dose may also comprisefrom about 1 μg/kg/body weight to about 1000 mg/kg/body weight (thissuch range includes intervening doses) or more per administration, andany range derivable therein. In non-limiting examples of a derivablerange from the numbers listed herein, a range of about 5 μg/kg/bodyweight to about 100 mg/kg/body weight, about 5 μg/kg/body weight toabout 500 mg/kg/body weight, etc., can be administered. The practitionerresponsible for administration will, in any event, determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject.

The active compounds can be formulated for parenteral administration,e.g., formulated for injection via the intravenous, intramuscular,sub-cutaneous, or even intraperitoneal routes. Typically, suchcompositions can be prepared as either liquid solutions or suspensions;solid forms suitable for use to prepare solutions or suspensions uponthe addition of a liquid prior to injection can also be prepared; and,the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The proteinaceous compositions may be formulated into a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

A pharmaceutical composition can include a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion, and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

B. Combination Treatments

In certain embodiments, the compositions and methods of the presentembodiments involve an antibody or an antibody fragment against HER3 toinhibit its activity in cancer cell proliferation, in combination with asecond or additional therapy. Such therapy can be applied in thetreatment of any disease that is associated with HER3-mediated cellproliferation. For example, the disease may be cancer.

The methods and compositions, including combination therapies, enhancethe therapeutic or protective effect, and/or increase the therapeuticeffect of another anti-cancer or anti-hyperproliferative therapy.Therapeutic and prophylactic methods and compositions can be provided ina combined amount effective to achieve the desired effect, such as thekilling of a cancer cell and/or the inhibition of cellularhyperproliferation. This process may involve contacting the cells withboth an antibody or antibody fragment and a second therapy. A tissue,tumor, or cell can be contacted with one or more compositions orpharmacological formulation(s) comprising one or more of the agents(i.e., antibody or antibody fragment or an anti-cancer agent), or bycontacting the tissue, tumor, and/or cell with two or more distinctcompositions or formulations, wherein one composition provides 1) anantibody or antibody fragment, 2) an anti-cancer agent, or 3) both anantibody or antibody fragment and an anti-cancer agent. Also, it iscontemplated that such a combination therapy can be used in conjunctionwith chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing, for example, both agents are delivered to a cellin a combined amount effective to kill the cell or prevent it fromdividing.

An inhibitory antibody may be administered before, during, after, or invarious combinations relative to an anti-cancer treatment. Theadministrations may be in intervals ranging from concurrently to minutesto days to weeks. In embodiments where the antibody or antibody fragmentis provided to a patient separately from an anti-cancer agent, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the two compounds wouldstill be able to exert an advantageously combined effect on the patient.In such instances, it is contemplated that one may provide a patientwith the antibody therapy and the anti-cancer therapy within about 12 to24 or 72 h of each other and, more particularly, within about 6-12 h ofeach other. In some situations it may be desirable to extend the timeperiod for treatment significantly where several days (2, 3, 4, 5, 6, or7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respectiveadministrations.

In certain embodiments, a course of treatment will last 1-90 days ormore (this such range includes intervening days). It is contemplatedthat one agent may be given on any day of day 1 to day 90 (this suchrange includes intervening days) or any combination thereof, and anotheragent is given on any day of day 1 to day 90 (this such range includesintervening days) or any combination thereof. Within a single day(24-hour period), the patient may be given one or multipleadministrations of the agent(s). Moreover, after a course of treatment,it is contemplated that there is a period of time at which noanti-cancer treatment is administered. This time period may last 1-7days, and/or 1-5 weeks, and/or 1-12 months or more (this such rangeincludes intervening days), depending on the condition of the patient,such as their prognosis, strength, health, etc. It is expected that thetreatment cycles would be repeated as necessary.

Various combinations may be employed. For the example below an antibodytherapy is “A” and an anti-cancer therapy is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of any compound or therapy of the present embodiments toa patient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the agents.Therefore, in some embodiments there is a step of monitoring toxicitythat is attributable to combination therapy.

i. Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance withthe present embodiments. The term “chemotherapy” refers to the use ofdrugs to treat cancer. A “chemotherapeutic agent” is used to connote acompound or composition that is administered in the treatment of cancer.These agents or drugs are categorized by their mode of activity within acell, for example, whether and at what stage they affect the cell cycle.Alternatively, an agent may be characterized based on its ability todirectly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such asthiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines, includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics, such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gammall andcalicheamicin omegall); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and angui dine); urethan; vindesine;dacarb azine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids, or derivatives of any of theabove.

ii. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated, such as microwaves, proton beamirradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), andUV-irradiation. It is most likely that all of these factors affect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

iii. Immunotherapy

The skilled artisan will understand that additional immunotherapies maybe used in combination or in conjunction with methods of theembodiments. In the context of cancer treatment, immunotherapeutics,generally, rely on the use of immune effector cells and molecules totarget and destroy cancer cells. Rituximab (Rituxan®) is such anexample. The immune effector may be, for example, an antibody specificfor some marker on the surface of a tumor cell. The antibody alone mayserve as an effector of therapy or it may recruit other cells toactually affect cell killing. The antibody also may be conjugated to adrug or toxin (chemotherapeutic, radionuclide, ricin A chain, choleratoxin, pertussis toxin, etc.) and serve merely as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target. Various effector cells include cytotoxic T cells and NKcells.

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present embodiments. Common tumormarkers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor,erb B, and p155. An alternative aspect of immunotherapy is to combineanticancer effects with immune stimulatory effects. Immune stimulatingmolecules also exist including: cytokines, such as IL-2, IL-4, IL-12,GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growthfactors, such as FLT3 ligand.

Examples of immunotherapies currently under investigation or in use areimmune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum,dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998);cytokine therapy, e.g., interferons α, β, and γ, IL-1, GM-CSF, and TNF(Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998);gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998;Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-gangliosideGM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat.No. 5,824,311). It is contemplated that one or more anti-cancertherapies may be employed with the antibody therapies described herein.

iv. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative, andpalliative surgery. Curative surgery includes resection in which all orpart of cancerous tissue is physically removed, excised, and/ordestroyed and may be used in conjunction with other therapies, such asthe treatment of the present embodiments, chemotherapy, radiotherapy,hormonal therapy, gene therapy, immunotherapy, and/or alternativetherapies.Tumor resection refers to physical removal of at least part ofa tumor. In addition to tumor resection, treatment by surgery includeslaser surgery, cryosurgery, electrosurgery, andmicroscopically-controlled surgery (Mohs' surgery).

Upon excision of part or all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection, or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

v. Other Agents

It is contemplated that other agents may be used in combination withcertain aspects of the present embodiments to improve the therapeuticefficacy of treatment. These additional agents include agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Increases inintercellular signaling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with certain aspectsof the present embodiments to improve the anti-hyperproliferativeefficacy of the treatments. Inhibitors of cell adhesion are contemplatedto improve the efficacy of the present embodiments. Examples of celladhesion inhibitors are focal adhesion kinase (FAKs) inhibitors andLovastatin. It is further contemplated that other agents that increasethe sensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with certain aspects of thepresent embodiments to improve the treatment efficacy.

III. Kits and Diagnostics

In various aspects of the embodiments, a kit is envisioned containingtherapeutic agents and/or other therapeutic and delivery agents. In someembodiments, the present embodiments contemplates a kit for preparingand/or administering a therapy of the embodiments. The kit may compriseone or more sealed vials containing any of the pharmaceuticalcompositions of the present embodiments. The kit may include, forexample, at least one HER3 antibody as well as reagents to prepare,formulate, and/or administer the components of the embodiments orperform one or more steps of the inventive methods. In some embodiments,the kit may also comprise a suitable container, which is a containerthat will not react with components of the kit, such as an eppendorftube, an assay plate, a syringe, a bottle, or a tube. The container maybe made from sterilizable materials such as plastic or glass.

The kit may further include an instruction sheet that outlines theprocedural steps of the methods set forth herein, and will followsubstantially the same procedures as described herein or are known tothose of ordinary skill in the art. The instruction information may bein a computer readable media containing machine-readable instructionsthat, when executed using a computer, cause the display of a real orvirtual procedure of delivering a pharmaceutically effective amount of atherapeutic agent.

IV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Generation of Rabbit Monoclonal Antibodies from a PhageDisplay Fab Library Using HER3 Antigen Binding Plasma B Cells Isolatedfrom Antigen Immunized Rabbit

ErbB3 (Genebank accession no. M34309) extracellular domain (ECD) (aminoacids Metl-Thr643) protein was used to immunize New Zealand rabbits atBethyl laboratories, Inc (Montgomery, Tex.). Rabbits were immunized with100 μg per rabbit dosing. After initial immunization, two boost dosingswere administered with a 2-3 week interval between dosings. The titer ofanti-Her3 sera was determined by a series of dilutions of serum in ELISAby coating HER3 ECD protein on 96-well plates (max-sorb plates, Nunc)and binding antibodies were detected with an anti-rabbit antibodyconjugated with horseradish peroxidase (HRP) and TMB substrate.Absorbance at 450 nm was used to determine the antibody serum titer.When the titer reached >10⁶, whole blood samples were withdrawn from theimmunized rabbits and plasma B cells (CD45+CD5-CD19+) were isolated fromthe freshly prepared rabbit peripheral blood mononuclear cells using afluorescence assisted cell sorting (FACS) instrument (BD FACSAria™ III,BD Biosciences). The isolated plasma cells were further selected forHER3 binding cells. After enrichment of antibody producing B cells,total RNA was isolated and cDNA was synthesized using a superscriptreverse transcriptase II (Invitrogen) according to manufacturer'ssuggestion. DNA sequences of antibody variable regions from both heavychains and light chains were amplified by polymerase chain reaction(PCR) using a set of designed primers (Table 1), see, e.g., Ridder etal., 2001, incorporated herein by reference. The amplified DNA wasconstructed into a phage display Fab expression vector (FIG. 2) andtransformed into E. coli (TG1) cells. A library size of 10⁸⁻⁹ wasconstructed in the phage display vector system and HER3 specific bindingFab was selected out through two rounds enrichment panning and sequenced(Lone Star Labs, Tex.). The process flow for antibody selection isillustrated in FIG. 1.

Selected HER3 binding hits were expressed as full length IgG in rabbitand rabbit/human chimeric forms using a mammalian expression vectorsystem in human embryonic kidney (HEK293) cells (Invitrogen) andpurified using a protein G resin with a fast protein liquidchromatography (FPLC) separation unit. Purified HER3 antibodies werecharacterized for various biological properties.

TABLE 1 PCR primer sets for cloning variable heavy and light chaincDNA sequences from rabbit plasma B cells Primer name Sequence RVκF1CGGCCTCTAGAGAGCTCGTGMTGACCCAGACT (SEQ ID NO: 41) RVκF2CGGCCTCTAGAGAGCTCGATMTGACCCAGACT (SEQ ID NO: 42) RVκR1GCATCCGTACGTCGTAGGATCTCCAGCTCGGT (SEQ ID NO: 43) RVκR2GCATCCGTACGTCGTTTGATTTCCACATTGGT (SEQ ID NO: 44) RVκR3GCATCCGTACGTCGTTTGACSACCACCTCGGT (SEQ ID NO: 45) RVλ FACAAGTCTAGAGAGCTCGTGCTGACTCAGTC (SEQ ID NO: 46) RVλ RCATCCGTACGTCGGCCTGTGACGGTCAGCT (SEQ ID NO: 47) RVH F1CTTCCCTCGAGCAGTCGGTGGAGGAGTCCRGG (SEQ ID NO: 48) RVH F2CTTCCCTCGAGCAGTCGGTGAAGGAGTCCGAG (SEQ ID NO: 49) RVH F3CTTCCCTCGAGCAGTCGYTGGAGGAGTCCGGG (SEQ ID NO: 50) RVH F4CTTCCCTCGAGCAGSAGCAGCTGRTGGAGTCC (SEQ ID NO: 51) RVH R1CATCGGGCCCTTGGTGGAGGCTGARGAGAYGGTGACCAGG (SEQ ID NO: 52)

Example 2 Measurements of Binding of Human and Mouse HER3/ErbB3 withFlow Cytometry and ELISA

CHO Flp-in (Invitrogen) cell lines expressing human or mouse HER3receptors were used to study binding of the HER3 antibodies on cellsurface receptors. HER3 expressing cells were detached using enzymefree-EDTA dissociation solution and resuspended in PBS, 2% FBS at aconcentration of approximately 1×10⁶ cells/ml. The cells were stainedwith the purified HER3 IgGs at 4° C. for 40 minutes and washed twice inPBS with 2% FBS by centrifugation at 1200 rpm for 10 minutes.Supernatant was removed and cells were stained with R-PE conjugatedanti-rabbit IgG at 4° C. for 30 min. Then cells were washed with 4 mLPBS with 2% FBS and were analyzed on a flow cytometer (Quava,Millipore). FIG. 3 shows the binding of the rabbit HER3 antibody onhuman and mouse HER3 expression cells as indicated by the increase offluorescence intensity (X-axis) in comparison with non-specific rabbitIgG binding control.

Example 3 Determination of HER3 Binding Affinity Using Surface PlasmonResonance (SPR) Assay with Biacore T-100

All experiments were performed at 25° C. at a flow rate of 45 μl/min. Toprepare a BIAcore assay, an anti-rabbit IgG antibody (50 μg/ml each inacetate buffer, pH 5.0) was immobilized onto a carboxymethyl dextransensorchip (CMS) using amine coupling procedures as described by themanufacturer. Purified rabbit Mabs to be tested were diluted at aconcentration of 5 μg/ml in 0.5% P20, HBS-EP buffer and injected on FC2to reach 500 to 1000 RU. FC1 was used as the reference cell. Specificsignals correspond to the difference of signals obtained on FC2 versusFC1. The analyte (recombinant human HER3, apparent molecular weight 97kDa on SDS-PAGE gel) was injected during 90 sec at series ofconcentration dilutions (100, 50, 25, 12.5, 6.25, and 3.13, 1.56 nM) in0.5% P20, HBS-EP buffer. These concentrations were prepared from stocksolution in 0.5% P20, HBS-EP. The dissociation phase of the analyte wasmonitored over a 30 minute period. Running buffer was also injectedunder the same conditions as a double reference. After each runningcycle, both flow cells were regenerated by injecting 20 to 45 μl ofglycine-HCl buffer pH 1.5. Binding K_(D) on HER3 was calculated bykoff/kon kinetic rate for each HER3 monoclonal antibody (Table 2).

TABLE 2 HER3 antibody binding kinetic constants on human HER3/ErbB3extracellular domain determined using BIAcore (SPR) analysis Purifiedmonoclonal HER3 antibody K_(D) (nM) Rab46 7.3 ± 0.13 Rab1210 1.0 ± 0.05Rab189 3.6 ± 0.16 Rab774 0.16 ± 0.01 

Example 4 Internalization Study of HER3 Monoclonal Antibodies

T47D breast cancer cells were cultured to 80% confluency. The cells weredetached and resuspended in complete culture medium for accountingcells. Cells at a concentration of approximately 1×10⁷/ml were incubatedat 37° C. for 3 hr in the presence and absence of HER3 IgG at 2 μg/ml.Then cells were washed with 4 mL PBS, 2% FBS and stained with the HER3antibody (10 μg/ml) as primary binding antibody at 4° C. for 40 minutes.After washing with PBS, 2% FBS, cells were stained with 0.5 μg R-PEconjugated goat anti-rabbit antibody (BD Pharmingen) at 4° C. for 30minutes, covered with foil. Cells were washed with 4 mL PBS, 2% FBS,then centrifuged at 1200 rpm for 10 minutes. Cells were fixed in PBS/10%formaldehyde and analyzed using a flow cytometer (Quava, Millipore).Percentage of HER3 receptor internalization mediated by the isolatedHER3 monoclonal antibodies was calculated by the formula: (meanfluorescence intensity (MFI) of no antibody control-MFI of antibodytreated cells)/MFI of controls×100, shown in Table 3.

TABLE 3 Percentage of HER3 receptor internalization mediated byanti-HER3 antibodies HER3 monoclonal antibody % Receptor InternalizationRab46 39 ± 5 Rab1210 62 ± 5 Rab189 55 ± 4 Rab774 60 ± 8

Example 5 Inhibition of pHER3, pAKT, and pERK1/2 by Rabbit HER3Monoclonal Antibodies

MCF7 cells were seeded at cell density approximately 3×10⁵ cells/ml in96-well plates and cultured over night. Cell culture media were changedto low serum (0.5% FBS) for 15 hours, cells were treated with theisolated HER3 monoclonal antibodies for 2 hr at 37° C. Cells werestimulated with 3.3 nM rhNRG1-β1 for 20 minutes at 37° C. before makingcell lysates. Before making cell lysates, cells were washed with coldPBS, 0.5% BSA, for 3 times. Cell Extraction Buffer (containing fresh 1mM PMSF, protease inhibitor cocktail, and phosphatase inhibitorcocktail) was added into each well for 30 min with gentle rocking at 4°C. After lysate was mixed by pipetting up-down for 3-5 times, the platewas centrifuged at 3, 000 rpm for 10 min and cell supernatants were usedin following assays.

For pHER3 inhibition assay, maxi-sorp plate (Costar 96 well plates) wascoated with 4 μg/ml mouse anti-human HER3 antibody (R&D Systems) at 4°C. for over night and was blocked with PBS containing 2% BSA for 2 hoursat room temperature. 100 μl of cell supernatant was transferred to theblocked assay plate and incubated for 2 hr at RT. Plates were washedwith 300 μl PBS with Tween 20 (0.05%) for 5 times. Then secondaryantibody, anti-p-tyrosine-HRP (R&D Systems), was added and incubated for1 hr at RT before detection. Washing steps were repeated and HRPchemiluminescence substrate (Millipore, Calif.) was added for 5 minuteswith gentle rocking at RT, then luminescent signals were read using aplate reader (Molecular Devices, CA). Percentage of inhibition of pHER3was calculated using the formula: (signals of cells without antibodytreatment-signals of antibody treated cells)/signals of controls×100.Average of 3-4 replications was used in the concentration dependentinhibition assay (FIG. 4). IC₅₀s determined using the titration curvefit by GraphPad prism (v5.1) are listed in the Table 4.

For pAKT inhibition assay, similar ELISA format was used for capturingtotal AKT proteins using an antibody from R&D Systems. After washing theplates with 300 μl/well PBS-T for 5 times and secondary antibody,biotinylated rabbit anti-human phospho-Akt (S473) was added for 1 hrincubation at RT. Streptavidin-HRP was used for detection by incubationfor 20 min at RT. After washing the plates, HRP chemiluminescencesubstrate was added for 5-10 minutes and plate was read using a platereader (Molecular Devices). Percentage of inhibition of pAKT wascalculated using the formula: (signals of cells without antibodytreatment-signals of antibody treated cells)/signals of controls×100.Average of 3-4 replications was used in the concentration dependentinhibition assay (FIG. 5). IC₅ s determined using the titration curvefit by GraphPad prism (v5.1) are listed in the Table 4.

For pERK1/2 inhibition assay, Meso Scale Discovery (MSD) pre-coatedplate was blocked with TRIS buffer containing 3% BSA with shaking. Celllysates (25 μl/well) were transferred to the blocked assay plate andincubated for 2 hr at RT with shaking. Wells were washed with 300μl/well Tris buffer for 3 times and detected with MSDanti-phospho-ERK1/2 (T/Y: 202/204; 185/187) antibody and SULFO-TAG™solution from MSD assay kit according to the manufacturer's suggestion.Plate was read on MSD SECTOR™ Imager 2400 and data was analyzed usingthe formula: (signals of cells without antibody treatment-signals ofantibody treated cells)/signals of controls×100. Average of 3-4replications was used in the concentration dependent inhibition assay(FIG. 5). IC₅₀ determined using the titration curve fit by GraphPadprism (v5.1) are listed in the Table 4.

Example 6 Blocking Ligand Neuregulin Binding on HER3 by the HER3Monoclonal Antibodies

HER3 ligand neuregulin (NRG1) can bind on HER3 extracellular domain andactivates HER3 phosphorylation and down-stream signaling. Blockingligand binding on HER3 by the selected rabbit HER3 monoclonal antibodieswas determined using alpha screen format assay. NRG1 (R&D Systems) wasbiotinylated using a biotinylation kit (Fisher Scientific) and HER3 ECDwas tagged with 10 histidine (His). Purified anti-HER3 Mabs wereserially diluted two-fold into an assay buffer made from 25 mM Hepes,100 mM NaCl, 0.5% BSA and 0.05% Tween20 in an opaque/white, half-wellarea plate (Costar). Rabbit IgG1 was used as a negative control.Biotinylated NRG-lbeta ligand and Her3/His₁₀ receptor were addedsequentially at 10 nM concentration to the assay plate containingserially diluted antibodies. The mixture of receptor, ligand and theantibodies was incubated at room temperature with gentle shaking for 90minutes. AlphaScreen Streptavidin Donor beads and His-Nickel Acceptorbeads were added into the assay at a final concentration of 20 μg/mleach. The assay plates were covered with foil and incubated at roomtemperature with gentle shaking for 1 hour. The plates were read inEnVision plate reader. Blocking of NRG1 ligand binding to HER3 by theHER3 monoclonal antibodies showed concentration dependence (FIG. 6) andIC₅₀ values (Table 4) were estimated using four parameter curve fittingwith concentration titration graphs using the GraphPad.

TABLE 4 IC50 estimated from dose dependence titration curves forinhibition of pHER3, inhibition of pAKT, inhibition of pERK, and NRGligand binding by anti-HER3 antibodies HER3 pHER3 pAKT pERK1/2 BlockingNRG monoclonal inhibition, inhibition, inhibition, binding, EC50antibody EC50 (nM) EC50 (nM) EC50 (nM) (nM) Rab46 3.5 3.8 6.2 0.51Rab1210 0.7 0.4 1.3 0.10 Rab189 1.7 3.6 5.5 0.07 Rab774 1.8 2.3 3.8 0.52

Example 7 Inhibition of Cancer Cell Proliferation by Anti-HER3 mAbs

In order to determine inhibition of cancer cell proliferation by theisolated HER3 antibodies, 3000 cells/well of MCF7 (human breast cancer)cancer cells were seeded in 100 μl of 10% FBS medium on 96-well a 37° C.incubator overnight. The medium was replaced with 0.5% FBS medium withHER3 antibodies and were cultured for 72 hours with stimulation with 100ng/ml β-NRG by adding the ligand directly to antibody containing medium.Cell proliferation was measured by adding 10 μl of AlamarBlum™(Invitrogen) added to the medium and the cells were incubated at 37° C.incubator and fluorescence signals were measured after two hours afteraddition of AlamarBlum™ at excitation 535 nm and emission at 590 nm.Inhibition of NRG-induced cell growth in MCF7 cells was calculated aspercentage of reduction by the antibodies relative to no antibodytreatment (FIG. 7) and IC₅₀ values are listed in Table 5.

TABLE 5 IC50 of inhibition of cancer cell proliferation by anti-HER3monoclonal antibodies HER3 monoclonal antibody IC50 (95% confidencelevel), nM Rab46 15.28 to 42.28 Rab1210 11.92 to 28.53 Rab189 31.66 to86.07 Rab774 151.2 to 526.7

Example 8 Sequencing Antibody Variable Region of DNA and Amino AcidSequences

IgG light chain variable region (LV) and heavy chain variable region(HV) were sequenced by Genewiz (Edison, N.J.). Both light chain variableand heavy chain variable amino acid sequences are listed Table 6. CDRsof light chain and heavy chain were analyzed using IMGT program and arelisted in Table 6.

TABLE 6 Anti-HER3 antibody sequences Heavy Chain SequenceLight Chain Sequence CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 mAbAmino acid sequence Amino acid sequence Rab46 GFSFSASY ITGDDDKR TRNLPDELESVYSYKN QAS (SEQ QGTYYSSA Y (SEQ T (SEQ (SEQ ID (SEQ ID ID NO: WDFVID NO: 9) ID NO: 10) NO: 11) NO: 12) 13) (SEQ ID NO: 14)EEQLVESGGGLVQPEGSLTLTCTASGF QAVVTQTPSSVSAAVGGTVTISCQSSESFSASYYIYWVRQAPGKGLEGIAYITG SVYSYKNLAWYQQKPGQPPRLLIWQASDDDKRTYYANWAKGRFTISKTSTTVTL RLSSGVSSRFSGSGSGTQFTLTISGVQQMTSLTAADTATYFCTRNLPDELWGPG CDDAATYYCQGTYYSSAWDFVFGGGTETLVTVSS (SEQ ID NO: 1) VMVE (SEQ ID NO: 2) Rab1210 GFSFSSSY IWSGNGATARNYDASG QSIVSSY SAS (SEQ LYGVDSSN W (SEQ (SEQ ID YGIFHL (SEQ IDID NO: 19) IDNA ID NO: 15) NO: 16) (SEQ ID NO: 18) (SEQ ID NO: 17)NO: 20) QEQLEESGGGLVQPEGSLTLTCTASGF QAVVMTQTPASVSAAVGGTVTINCQASSFSSSYWICWVRQAPGKGLELIGYIWS QSIVSSYLSWYQQKTGQPPKLLIYSASGNGATYYASWAKGRFTISKTSSTTVTL KLASGVPPRFSGSGSGTQFTLTISGVQQMTSVTATDTATYFCARNYDASGYGIF CDDAATYYCLYGVDSSNIDNAFGGGTEHLWGPGTLVTVSS (SEQ ID NO: 3) VVVK (SEQ ID NO: 4) Rab189 GIGFGYYYIYTGISDS ARSDPYYT QSINNY RAS (SEQ QSYYYSSS Y (SEQ T (SEQ TTYTNTYP(SEQ ID ID NO: 25) NNYYNA ID NO: 21) ID NO: 22) TYWNL NO: 24) (SEQ ID(SEQ ID NO: 26) NO: 23) QESLEESGGDLVKPEGTLTLICKASGICAFELTQTPSSVEAAVGGTVTIKCQAS GFGYYYYICWVRQAPGKGLEWIACIYTQSINNYLAWYQQKTGQPPKLLIYRAST GISDSTYYATWAKGRFAISKTSSTTVTLESGVPSRFKGSGSGTQFTLTISDLEC LQMTSLTAADTATYFCARSDPYYTTTYADAATYYCQSYYYSSSNNYYNAFGGGT TNTYPTYWNLWGPGTLVTVSS (SEQEVVVK (SEQ ID NO: 6) ID NO: 5) Rab774 GIDLSTYA INRSSKT GRVMSPYS SDLSDYTLRSDGSY GLPYSVEY (SEQ ID (SEQ ID VAGGDL (SEQ ID (SEQ ID V (SEQ NO: 27)NO: 28) (SEQ ID NO: 30) NO: 31) ID NO: NO: 29) 32)QSVEESGGRLVTPGTPLTLTCTVSGID QPVLTQSPSASAALGASAKLTCILSSDLSTYAMTWVRRAPGKGLEWLGIINRSS LSDYTIDWYKHQQGEAPRYLMQLRSDGKTYLTDWARGRFTISKTSTTVDLKISS SYTKGTGVPDRFSGSSSGADRYLVISSPTTEDTATYFCGRVMSPYSVAGGDLWG VQADDEADYYCGLPYSVEYVFGGGTQLPGTLVTVSS (SEQ ID NO: 7) TVT (SEQ ID NO: 8)

Example 9 Humanization of Rabbit HER3 Monoclonal Antibodies

The best matched human VH and VL germline sequences to the rabbit VH andVL sequences were identified using the IMGT data base (Lafranc et al.,2012, incorporated herein by reference). Humanization was based on theCDR grafting concept (Haidar et al., 2012) and CDRs of the rabbitantibodies were defined by the combination of Kabat/Chothia, Yu et al.,2010 and Haidar et al. 2012 (See also Retter et al., 2005 and Singer etal., 1993).

Total of 2 heavy chains and 2 light chains of variable sequences weredesigned for each of anti-HER3 monoclonal antibodies: Rab46 and Rab1210as shown below in Chart 1 (CDR sequences are underlined).

CHART 1 Variable domains of humanized antibodies. >hRab46H-1QVQLVESGGGLVQPGGSLRLSCSASGFSFSASYYIYWVRQAPGKGLEYIAYITGDDDKRTYYANWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCTRNLPDELWGPGTLVTVSS (SEQ ID NO: 33) >hRab46H-2QVQLVESGGGLVQPGGSLRLSCSASGFSFSASYYIYWVRQAPGKGLEYIAYITGDDDKRTYYANWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCTRNLPDELWGPGTLVTVSS (SEQ ID NO: 34) >hRab46L-1DIQMTQTPSTLSASVGDRVTITCQSSESVYSYKNLAWYQQKPGKPPKLLIWQASRLSSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGTYYSSAWDFVFGGGTKVEIK (SEQ ID NO: 35) >hRab46L-2QAQMTQTPSTLSASVGDRVTITCQSSESVYSYKNLAWYQQKPGKPPKLLIWQASRLSSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGTYYSSAWDFVFGGGTKVEIK (SEQ ID NO: 36) >hRab1210H-1QVQLVESGGGLVQPGGSLRLSCSASGFSFSSSYWICWVRQAPGKGLEYIGYIWSGNGATYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARNYDASGYGIFHLWGPGTLVTVSS (SEQ ID NO: 37) >hRab1210H-2QVQLVESGGGLVQPGGSLRLSCSASGFSFSSSYWICWVRQAPGKGLELIGYIWSGNGATYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYFCARNYDASGYGIFHLWGPGTLVTVSS (SEQ ID NO: 38) >hRab1210L-1DIQMTQTPSSLSASVGDRVTINCQASQSIVSSYLSWYQQKPGKPPKLLIYSASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLYGVDSSNIDNAFGGGTKVEIK (SEQ ID NO: 39) >hRab1210L-2AVQMTQTPSSLSASVGDRVTINCQASQSIVSSYLSWYQQKPGKPPKLLIYSASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLYGVDSSNIDNAFGGGTKVEIK (SEQ ID NO: 40)

Construction and Production of Humanized Monoclonal Antibodies

The DNA sequences from the designed humanized variable regions weresynthesized and cloned into IgG1 expression vectors as described in theprevious examples for production of the humanized antibodies in HEK293cells. Total of 8 humanized monoclonal antibodies were produced byrecombination of 1 heavy chain and 1 light chain in each of antibodies.The composition of heavy and light chains for each of humanizedantibodies is listed in the Table 8 below.

TABLE 8 Heavy and light chain variable regions of the humanized HER3mAb. Name of Parental Heavy Chain Light Chain Humanized Clone VariableVariable Antibody Name Sequences Sequences HER3-hMab-A9 Rab46 hRab46H-1hRab46L-1 HER3-hMab-A10 Rab46 hRab46H-2 hRab46L-1 HER3-hMab-A11 Rab46hRab46H-1 hRab46L-2 HER3-hMab-A12 Rab46 hRab46H-2 hRab46L-2HER3-hMab-A13 Rab1210 hRab1210H-1 hRab1210L-1 HER3-hMab-A14 Rab1210hRab1210H-2 hRab1210L-1 HER3-hMab-A15 Rab1210 hRab1210H-1 hRab1210L-2HER3-hMab-A16 Rab1210 hRab1210H-2 hRab1210L-2

Characterization of Humanized Anti-HER3 Monoclonal Antibodies

Binding of the humanized HER3 monoclonal antibodies on extracellulardomain (ECD) of HER3 by ELISA: All monoclonal antibodies showed strongbinding on human HER3 ECD similar to the parental rabbit monoclonalantibodies, as shown in FIG. 8A. For the panel of 4 humanized antibodiesfrom the parental Rab46, all 4 antibodies retained binding affinity onmouse HER3 ECD domains (FIG. 8B). The estimated EC50s (concentrationsgive 50% of maximum binding capacity) for those humanized antibodies arelisted in Table 9.

TABLE 9 EC₅₀s (μg/ml) of humanized HER3 antibodies by ELISA. HER3Antigen A9 A10 A11 A12 A13 A14 A15 A16 Chi46 Chi1210 Human HER3 0.00310.0026 0.0026 0.0024 0.0025 0.0019 0.0024 0.0023 0.0027 0.0026 ECD MouseHER3 0.0043 0.0054 0.0079 0.0067 0.0059 ECD

HER3 antigen binding kinetics of the humanized HER3 antibodies measuredby surface Plasmon resonance (SPR) method: Kinetic constants of theantibodies were determined using a T-100 Biacore instrument (Table 10).SPR sensorgrams of the humanized HER3 antibodies and their parentalrabbit/human Fc chimeras are shown in FIG. 9.

TABLE 10 Kinetic binding constants for humanized HER3 antibodies.Antibody ka (1/Ms) kd (1/s) KD (M) A10 1.41E+05 1.69E−04 1.20E−09 A142.01E+05 1.74E−04 8.62E−10 CHI46 1.38E+05 4.58E−04 3.32E−09 CHI12105.38E+05 6.44E−04 1.20E−09

Inhibition of HER3 phosphorylation by humanized HER3 monoclonalantibodies: Assay of HER3 signaling inhibition was conducted asdescribed for rabbit HER3 antibodies in the earlier section. As shown inFIG. 10, the humanized HER3 antibodies, A10 and A14, showed equal tobetter inhibition of HER3 phosphorylation than their parentalantibodies. Estimated IC₅₀ of pHER3 inhibition from the concentrationtitration graphs (FIG. 10) is shown in Table 11.

TABLE 11 Estimated IC₅₀ for inhibition of pHER3 by the humanized HER3antibodies. Antibody Name A10 A14 Chi46 Chi1210 pHER3 inhibition 0.01730.0017 0.0330 0.0035 (IC₅₀, μg/ml)

Inhibition of cancer cell proliferation by the humanized HER3 monoclonalantibodies: Inhibition of cancer cell proliferation was determined usingthe same procedures as described for studies with the rabbit monoclonalantibodies. Two cancer cell lines were used, one is breast cancer cellline MCF-7 and the other cell line is the prostate cancer cell lineCWR22, and both cell lines were from the ATCC (American Tissue CultureCollection). Inhibition of cancer cell proliferations showed positivecorrelation with the increased antibody concentrations as shown in FIGS.11 and 12. The estimated IC₅₀ for inhibition of cancer cellproliferation is shown in Table 12.

TABLE 5 Estimated IC₅₀ for inhibition of cancer cell proliferation bythe humanized HER3 antibodies. Antibody Name A10 A14 Chi46 Chi1210Inhibition of MCF-7 0.181 0.232 ND* 0.084 cancer cell proliferation(IC₅₀, μg/ml) Inhibition of CWR22 0.036 0.025 0.138 0.003 cancer cellproliferation (IC₅₀, μg/ml) *ND, not determined

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method for treating a subject having aHER3-positive cancer comprising administering an effective amount of anantibody to the subject, wherein the antibody comprises: (i) a V_(H)domain at least about 80% identical to the V_(H) domain of Rab46 (SEQ IDNO: 1); hRab46H-1 (SEQ ID NO: 33); or hRab46H-2 (SEQ ID NO: 34), whereinsaid V_(H) domain comprises CDRs 1-3 of SEQ ID NOS: 9-11; and a V_(L)domain at least about 80% identical to the V_(L) domain of Rab46 (SEQ IDNO: 2); hRab46L-1 (SEQ ID NO: 35); or hRab46L-2 (SEQ ID NO: 36), whereinsaid VL domain comprises CDRs 1-3 of SEQ ID NOS: 12-14; (ii) a V_(H)domain at least about 80% identical to the V_(H) domain of Rab1210 (SEQID NO: 3); hRab1210H-1 (SEQ ID NO: 37); or hRab1210H-2 (SEQ ID NO: 38),wherein said V_(H) domain comprises CDRs 1-3 of SEQ ID NOS: 15-17; and aV_(L) domain at least about 80% identical to the V_(L) domain of Rab1210(SEQ ID NO: 4); hRab1210L-1 (SEQ ID NO: 39); or hRab1210L-2 (SEQ ID NO:40), wherein said V_(L) domain comprises CDRs 1-3 of SEQ ID NOS: 18-20;(iii) a V_(H) domain at least about 80% identical to the V_(H) domain ofRab189 (SEQ ID NO: 5), wherein said V_(H) domain comprises CDRs 1-3 ofSEQ ID NOS: 21-23; and a V_(L) domain at least about 80% identical tothe V_(L) domain of Rab189 (SEQ ID NO: 6), wherein said V_(L) domaincomprises CDRs 1-3 of SEQ ID NOS: 24-26; or (iv) a V_(H) domain at leastabout 80% identical to the V_(H) domain of Rab774 (SEQ ID NO: 7),wherein said V_(H) domain comprises CDRs 1-3 of SEQ ID NOS: 27-29; and aV_(L) domain at least about 80% identical to the V_(L) domain of Rab774(SEQ ID NO: 8), wherein said V_(L) domain comprises CDRs 1-3 of SEQ IDNOS: 30-32.
 2. The method of claim 1, wherein the HER3-postive cancer isa breast cancer, lung cancer, head & neck cancer, prostate cancer,esophageal cancer, tracheal cancer, skin cancer brain cancer, livercancer, bladder cancer, stomach cancer, pancreatic cancer, ovariancancer, uterine cancer, cervical cancer, testicular cancer, coloncancer, rectal cancer or skin cancer.
 3. The method of claim 1, whereinthe antibody is in a pharmaceutically acceptable composition.
 4. Themethod of claim 1, wherein the antibody is administered systemically. 5.The method of claim 1, wherein the antibody is administeredintravenously, intradermally, intratumorally, intramuscularly,intraperitoneally, subcutaneously, or locally.
 6. The method of claim 1,further comprising administering at least a second anticancer therapy tothe subject.
 7. The method of claim 6, wherein the second anticancertherapy is a surgical therapy, chemotherapy, radiation therapy,cryotherapy, hormonal therapy, immunotherapy or cytokine therapy.
 8. Themethod of claim 1, wherein the antibody comprises a V_(H) domain atleast 95% identical to the V_(H) domain of hRab46H-1 (SEQ ID NO: 33) anda V_(L) domain at least 95% identical to the V_(L) domain of hRab46L-1(SEQ ID NO: 35) or hRab46L-2 (SEQ ID NO: 36).
 9. The method of claim 8,wherein the antibody comprises a V_(H) domain identical to the V_(H)domain of hRab46H-1 (SEQ ID NO: 33) and a V_(L) domain identical to theV_(L) domain of hRab46L-1 (SEQ ID NO: 35).
 10. The method of claim 8,wherein the antibody comprises a V_(H) domain identical to the V_(H)domain of hRab46H-1 (SEQ ID NO: 33) and a V_(L) domain identical to theV_(L) domain hRab46L-2 (SEQ ID NO: 36).
 11. The method of claim 1,wherein the antibody comprises a V_(H) domain at least 95% identical tothe V_(H) domain of hRab46H-2 (SEQ ID NO: 34) and a V_(L) domain atleast 95% identical to the V_(L) domain of hRab46L-2 (SEQ ID NO: 36) orhRab46L-1 (SEQ ID NO: 35).
 12. The method of claim 11, wherein theantibody comprises a V_(H) domain identical to the V_(H) domain ofhRab46H-2 (SEQ ID NO: 34) and a V_(L) domain identical to the V_(L)domain of hRab46L-2 (SEQ ID NO: 36).
 13. The method of claim 11, whereinthe antibody comprises a V_(H) domain identical to the V_(H) domain ofhRab46H-2 (SEQ ID NO: 34) and a V_(L) domain identical to the V_(L)domain of hRab46L-1 (SEQ ID NO: 35).
 14. The method of claim 1, whereinthe antibody comprises a V_(H) domain at least 95% identical to theV_(H) domain of hRab1210H-1 (SEQ ID NO: 37) and a V_(L) domain at least95% identical to the V_(L) domain of hRab1210L-1 (SEQ ID NO: 39) orhRab1210L-2 (SEQ ID NO: 40).
 15. The method of claim 14, wherein theantibody comprises a V_(H) domain identical to the V_(H) domain ofhRab1210H-1 (SEQ ID NO: 37) and a V_(L) domain identical to the V_(L)domain of hRab1210L-1 (SEQ ID NO: 39).
 16. The method of claim 14,wherein the antibody comprises a V_(H) domain identical to the V_(H)domain of hRab1210H-1 (SEQ ID NO: 37) and a V_(L) domain identical tothe V_(L) domain of hRab1210L-2 (SEQ ID NO: 40).
 17. The method of claim1, wherein the antibody comprises a V_(H) domain at least 95% identicalto the V_(H) domain of hRab1210H-2 (SEQ ID NO: 38) and a V_(L) domain atleast 95% identical to the V_(L) domain of hRab1210L-2 (SEQ ID NO: 40)or hRab1210L-1 (SEQ ID NO: 39).
 18. The method of claim 17, wherein theantibody comprises a V_(H) domain identical to the V_(H) domain ofhRab1210H-2 (SEQ ID NO: 38) and a V_(L) domain identical to the V_(L)domain of hRab1210L-2 (SEQ ID NO: 40).
 19. The method of claim 17,wherein the antibody comprises a V_(H) domain identical to the V_(H)domain of hRab1210H-2 (SEQ ID NO: 38) and a V_(L) domain identical tothe V_(L) domain of hRab1210L-1 (SEQ ID NO: 39).
 20. The method of claim17, wherein the antibody is the HER3-hMab-A9, HER3-hMab-A10,HER3-hMab-A11, HER3-hMab-A12, HER3-hMab-A13, HER3-hMab-A14,HER3-hMab-A15 or HER3-hMab-A16 antibody.